Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures

Abstract

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16-1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M1 replication defects of mak16-1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade 1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1.2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20 degrees C, 30 degrees C, or 37 degrees C, but could not grow at 8 degrees C. The deleted region is thus essential only at 8 degrees C, and we name this gene LTE1 (low temperature essential).

MOB1, an Essential Yeast Gene Required for Completion of Mitosis and Maintenance of Ploidy

“…The late nuclear division arrest phenotype of mob1 mutants is similar to that described for cells harboring mutations in a number of genes, including CDC5, CDC14, CDC15, and LTE1 (Pringle and Hartwell, 1981;Wickner et al, 1987). CDC5 and CDC15 encode essential protein kinases, CDC14 encodes an essential protein tyrosine phosphatase, and LTE1 encodes a GDP/ GTP exchange protein (Schweitzer and Philippsen, 1991;Wan et al, 1992;Kitada et al, 1993;Keng et al, 1994).…”

“…There are a number of genes, encoding regulatory proteins, that, when mutated, cause a cell cycle arrest similar to mob1 mutants. There is an extensive array of genetic interactions linking them together, suggesting that they belong to a common pathway (Pringle and Hartwell, 1981;Wickner et al, 1987;Johnston et al, 1990;Parkes and Johnston, 1992;Kitada et al, 1993;Molero et al, 1993;Spevak et al, 1993;Keng et al, 1994;Shirayama et al, 1994aShirayama et al, , 1994bShirayama et al, , 1996Toyn and Johnston, 1994). We have begun to establish that MOB1 shares genetic interactions with several members of this class of genes.…”

Global Screening of Genes Essential for Growth in High-Pressure and Cold Environments: Searching for Basic Adaptive Strategies Using a Yeast Deletion Library

“…Lte1 is a putative guanine nucleotide exchange factor required for mitotic exit at low temperature (Wickner et al 1987;Shirayama et al 1994a,b). Lte1 is localized to the bud cortex and bud cytoplasm from the S phase to M phase and is uniformly distributed in the G 1 phase (Yoshida et al 2003).…”

The RSF1 gene regulates septum formation in Saccharomyces cerevisiae

“…Further genetic analysis localized rsfl-J to the left arm of chromosome I (rsfl-J x adel, 11 PD:1 NPD:19 TT = 42 cM; rsfl-J x cysi, 6 PD:0 NPD:8 TT = 29 cM). No other mutation conferring a related phenotype has been genetically mapped to this area of chromosome I. Cloned DNA fragments of the left arm of chromosome I (20,33), which correspond to the genetic locale for rsfl-J, were tested for complementation of the rsfl-J phenotype. Mutant cells transformed with these fragments still expressed the rsfl-l phenotype (data not shown), indicating that RSFJ is distinct from the MAK16, LTEJ, FUN12, FUNI9, FUN20, FUN21, and FUN22 genes.…”